Identification of candidate circulating cisplatin-resistant biomarkers from epithelial ovarian carcinoma cell secretomes.

BACKGROUND: The majority of patients diagnosed with advanced epithelial ovarian carcinoma (EOC) relapse with resistant disease, and there are no biomarkers that possess clinical utility to identify or monitor these patients. This study aimed to identify secreted proteins ('secretome') collected from human EOC cell lines that differ in their inherent platinum sensitivity. METHODS: Secreted proteins collected from conditioned medium from ovarian cancer cell lines that vary in their sensitivity to cisplatin were digested with trypsin and analysed by liquid chromatography-tandem mass spectrometry for peptide identification. RESULTS: Of the 1688 proteins identified, 16 possessed significant differential abundances (P<0.05) between the platinum-resistant and -sensitive cell lines. A number of these were verified by immunoblot, including COL11A1, which was also found to be associated with worse progression-free survival (PFS; N=723) and overall survival (OS; N=1183) as assessed from publicly available transcript expression data from ovarian cancer tumour specimens. CONCLUSION: Secretome proteomics of EOC cells resulted in the identification of a novel candidate biomarker, COL11A1. The expression level of COL11A1 correlates to worse PFS and OS, and is predicted to reside in peripheral circulation making this an attractive candidate for validation in sera as a biomarker of cisplatin resistance and poor outcome.

BRCA1 expression and improved survival in ovarian cancer patients treated with intraperitoneal cisplatin and paclitaxel: a Gynecologic Oncology Group Study.

BACKGROUND: Breast cancer 1, early onset (BRCA1) is a tumour-suppressor gene associated with familial epithelial ovarian cancer (EOC). Reduced BRCA1 expression is associated with enhanced sensitivity to platinum-based chemotherapy. We sought to examine the prognostic relevance of BRCA1 expression in EOC patients treated with intraperitoneal platinum/taxane. METHODS: The GOG-172 was a phase III, multi-institutional randomised trial of intravenous paclitaxel and cisplatin (IV therapy) vs intravenous paclitaxel, intraperitoneal cisplatin plus paclitaxel (IP therapy) in patients with optimally resected stage III EOC. The BRCA1 expression was assessed with immunohistochemistry (IHC) staining blinded to clinical outcome in archival tumour specimens. Slides with 10% staining were defined as aberrant and >10% as normal. Correlations between BRCA1 expression and progression-free survival (PFS) and overall survival (OS) were analysed using Kaplan-Meier method and Cox regression analysis. RESULTS: Of the 393 patients, 189 tumours had aberrant expression, and 204 had normal BRCA1 expression. There was an interaction between BRCA1 expression and route of administration on OS (P=0.014) but not PFS (P=0.054). In tumours with normal BRCA1 expression, the median OS was 58 months for IP group vs 50 months for IV group (P=0.818). In tumours with aberrant BRCA1 expression, the median OS was 84 vs 47 months in the IP vs IV group, respectively (P=0.0002). Aberrant BRCA1 expression was an independent prognostic factor for better survival in women randomised to IP therapy (hazard ratio (HR)=0.67, 95% confidence interval (CI)=0.47-0.97, P=0.032). Similar survival was observed in the IV and IP patients with normal BRCA1 expression. Multivariate but not univariate modelling demonstrated that IV patients with aberrant vs normal BRCA1 expression had worse survival. CONCLUSION: Decreased BRCA1 expression is associated with a 36-month survival improvement in patients with EOC treated with IP chemotherapy. Although these results merit validation in future studies, the results suggest that decreased BRCA1 expression predicts for improved response to cisplatin-based IP chemotherapy with cisplatin and paclitaxel.

Endocervical glandular neoplasia associated with lobular endocervical glandular hyperplasia is HPV-independent and correlates with carbonic anhydrase-IX expression: a Gynaecological Oncology Group Study.

BACKGROUND: Lobular endocervical glandular hyperplasia (LEGH) is a rare lesion of the uterine cervix. It has been proposed that LEGH may represent a precursor lesion to a group of mucinous adenocarcinoma with gastric phenotype (GA) that is independent of high-risk human papillomavirus (H-HPV) infection. Carbonic anhydrase-IX (CA-IX) is highly expressed in conventional glandular lesions (CGLs). However, expression of CA-IX in LEGH or GA has not been studied. METHODS: In all, 12 CGLs, 7 LEGHs, 6 LEGHs with coexisting adenocarcinoma in situ (AIS, 3) and GA (3) were identified from Japanese women with a cytological diagnosis of atypical glandular cells of undetermined significance. Immunostaining was used to detect CA-IX and p16(INK)4(a) (hereafter termed p16) protein expression in the tissues and CA-IX protein expression in the Papanicolaou smears (PSs). Polymerase chain reaction was used to detect H-HPV DNA in liquid-based cytology. RESULTS: Out of 12 (83%) CGLs, 10 were positive with H-HPV and high levels of CA-IX expression were seen in all (100%) cases. P16 protein expression was observed in 11 out of 12 (92%) cases. None of the LEGHs, LEGHs with AIS or GA were positive for H-HPV and only 8 out of 13 (62%) showed focal weak (1+) p16 expression. In contrast, all cases (100%) exhibited strong CA-IX protein expression. CONCLUSION: Our study suggests that there are different molecular mechanisms of carcinogenesis resulting in CGLs vs LEGHs associated with AIS or GA. There is also a possible link between LEGHs and GAs. Furthermore, CA-IX expression may serve as a useful biomarker for the detection of GAs in the absence of H-HPV infection.

Knockdown of splicing factor SRp20 causes apoptosis in ovarian cancer cells and its expression is associated with malignancy of epithelial ovarian cancer.

Our previous study revealed that two splicing factors, polypyrimidine tract-binding protein (PTB) and SRp20, were upregulated in epithelial ovarian cancer (EOC) and knockdown of PTB expression inhibited ovarian tumor cell growth and transformation properties. In this report, we show that knockdown of SRp20 expression in ovarian cancer cells also causes substantial inhibition of tumor cell growth and colony formation in soft agar and the extent of such inhibition appeared to correlate with the extent of suppression of SRp20. Massive knockdown of SRp20 expression triggered remarkable apoptosis in these cells. These results suggest that overexpression of SRp20 is required for ovarian tumor cell growth and survival. Immunohistochemical staining for PTB and SRp20 of two specialized tissue microarrays, one containing benign ovarian tumors, borderline/low malignant potential (LMP) ovarian tumors as well as invasive EOC and the other containing invasive EOC ranging from stage I to stage IV disease, reveals that PTB and SRp20 are both expressed differentially between benign tumors and invasive EOC, and between borderline/LMP tumors and invasive EOC. There were more all-negative or mixed staining cases (at least two evaluable section cores per case) in benign tumors than in invasive EOC, whereas there were more all-positive staining cases in invasive EOC than in the other two disease classifications. Among invasive EOC, the majority of cases were stained all positive for both PTB and SRp20, and there were no significant differences in average staining or frequency of positive cancer cells between any of the tumor stages. Therefore, the expression of PTB and SRp20 is associated with malignancy of ovarian tumors but not with stage of invasive EOC.

Carbonic anhydrase IX (CA-IX) and high-risk human papillomavirus (H-HPV) as diagnostic biomarkers of cervical dysplasia/neoplasia in Japanese women with a cytologic diagnosis of atypical glandular cells (AGC): a Gynecologic Oncology Group (GOG) Study.

BACKGROUND: High-risk human papillomavirus (H-HPV) infection is linked to cervical neoplasia but its role in detecting cervical glandular lesions (GLs) is unclear. Carbonic anhydrase IX (CA-IX) is a hypoxic biomarker that is highly expressed in neoplastic cervical GLs. The diagnostic utility of these biomarkers was evaluated by the Gynecologic Oncology Group in Japanese women with a cytological diagnosis of atypical glandular cells. METHODS: Immunostaining was used to detect CA-IX in a conventional Pap smear. Immunoreactivity of CA-IX was interpreted by a panel of pathologists blinded to the histological diagnosis. Polymerase chain reaction was used to detect H-HPV in a liquid-based cytology specimen. RESULTS: Significant cervical lesions (SCLs), defined as cervical intraepithelial neoplasia (CIN2, CIN3), adenocarcinoma in situ or invasive carcinoma, were observed in 37/88 (42%) of women. CA-IX testing alone (n=88) had a sensitivity of 89, 100 or 73% for SCLs, GLs or significant squamous lesions (SLs), respectively, with a false negative rate (FNR) of 14%. Testing for H-HPV (n=84) had a sensitivity of 65, 53 or 80% for SCLs, GLs or SLs, respectively, with a FNR of 22%. The combination of CA-IX and H-HPV testing had a sensitivity of 97, 100 or 93% for SCLs, GLs or SLs, respectively, with a FNR of 5%. Among eight H-HPV-negative GLs, six (75%) had a diagnosis of lobular endocervical glandular hyperplasia (LEGH). CONCLUSION: The combination of CA-IX and HPV testing improved the diagnostic accuracy. The low rate of H-HPV positivity in the GLs was associated with coexisting LEGH independent of H-HPV.

Knockdown of polypyrimidine tract-binding protein suppresses ovarian tumor cell growth and invasiveness in vitro.

Polypyrimidine tract-binding protein (PTB) is an RNA-binding protein with multiple functions in the regulation of RNA processing and IRES-mediated translation. We report here overexpression of PTB in a majority of epithelial ovarian tumors revealed by immunoblotting and tissue microarray (TMA) staining. By western blotting, we found that PTB was overexpressed in 17 out of 19 ovarian tumor specimens compared to their matched-normal tissues. By TMA staining, we found PTB expression in 38 out of 44 ovarian cancer cases but only in two out of nine normal adjacent tissues. PTB is also overexpressed in SV40 large T-antigen immortalized ovarian epithelial cells compared to normal human ovarian epithelial cells. Using doxycycline-inducible small interfering RNA technology, we found that knockdown of PTB expression in the ovarian tumor cell line A2780 substantially impaired tumor cell proliferation, anchorage-independent growth and in vitro invasiveness. These results suggest that overexpression of PTB is an important component of the multistep process of tumorigenesis, and might be required for the development and maintenance of epithelial ovarian tumors. Moreover, because of its novel role in tumor cell growth and invasiveness, shown here for the first time, PTB may be a novel therapeutic target in the treatment of ovarian cancer.

Altered expression and localization of PKC eta in human breast tumors.

Protein kinase C (PKC) eta is a PKC isoform whose upregulation is associated with differentiation in many epithelial tissues, including the rat mammary gland. The purpose of this study was to examine whether PKC eta is altered, in expression or localization, in human breast cancer. Paraffin sections of 49 in situ breast lesions, 29 invasive breast tumors, and nine normal breast biopsies were examined for PKC eta expression by immunohistochemistry. Adjacent regions of normal epithelium, and in situ lesions that were present adjacent to invasive lesions were also analyzed. In normal epithelium, regardless of the presence of adjacent in situ or invasive lesions, PKC eta was present in the cytoplasm of the luminal epithelium, and increased in areas of normal lobular development, similar to normal rat mammary gland. PKC eta staining intensity was homogeneous in normal lobules, but heterogeneous in in situ and invasive lesions, being focally increased in cells with aberrant nuclear morphology. In situ lesions were similar to adjacent normal epithelium in average staining intensity, regardless of whether invasion was also present. However, the invasive lesions themselves were significantly decreased in staining intensity compared to adjacent in situ lesions. In addition, 75% of invasive breast cancer lesions showed decreased staining relative to adjacent normal epithelium, compared to 37% of in situ lesions. The invasive tumors which possessed high PKC eta staining were associated with positive lymph node status. These results demonstrate that quantitative and qualitative alterations in PKC eta occur in human breast cancers.

Changes in ErbB2 (her-2/neu), ErbB3, and ErbB4 during growth, differentiation, and apoptosis of normal rat mammary epithelial cells.

Studies were undertaken to examine the natural role of ErbB2, ErbB3, and ErbB4 during the development of normal rat mammary epithelial cells (MECs) in vivo and in vitro. Immunohistochemical analysis demonstrated that mammary gland terminal end buds expressed abundant ErbB2 and ErbB4 but limited ErbB3 in pubescent rats, whereas luminal epithelial cells in nulliparous rats expressed ErbB2, ErbB3, and/or ErbB4. During pregnancy, ductal epithelial cells and stromal cells expressed abundant ErbB3 but limited ErbB2. Although ErbB2 and ErbB3 were downregulated throughout lactation, both receptors were re-expressed during involution. In contrast, ErbB4 was downregulated throughout pregnancy, lactation, and involution. Immunoblotting and immunoprecipitation studies confirmed the developmental expression of ErbB2 and ErbB3 in the mammary gland and the co-localization of distinct ErbB receptors in the mammary gland of nulliparous rats. In agreement with our in vivo findings, primary culture studies demonstrated that ErbB2 and ErbB3 were expressed in functionally immature, terminally differentiated and apoptotic MECs, and downregulated in functionally differentiated MECs. ErbB receptor signaling was required for epithelial cell growth, functional differentiation, and morphogenesis of immature MECs, and the survival of terminally differentiated MECs. Finally, ErbB4 expression did not interfere with functional differentiation and apoptosis of normal MECs.

A developmental atlas of rat mammary gland histology.

The mammary gland is a dynamic tissue that undergoes epithelial expansion and invasion during puberty and cycles of branching and lobular morphogenesis, secretory differentiation, and regression during pregnancy, lactation, and involution. The alteration in the mammary gland epithelium during its postnatal differentiation is accompanied by changes in the multiple stromal cell types present in this complex tissue. The postnatal plasticity of the epithelium, endothelium, and stromal cells of the mammary gland may contribute to its susceptibility to carcinogenesis. The purpose of this review is to assist researchers in recognizing histological changes in the epithelium and stroma of the rat mammary gland throughout development.

Mammary fibroblasts stimulate growth, alveolar morphogenesis, and functional differentiation of normal rat mammary epithelial cells.

Stromal-epithelial interactions play a profound role in regulating normal and tumor development in the mammary gland. The molecular details of these events, however, are incompletely understood. A novel serum-free transwell coculture system was developed to study the natural paracrine interactions between mammary epithelial cells (MEC) and mammary fibroblasts (MFC) isolated from normal rats during puberty. The MEC were cultured within a reconstituted basement membrane (RBM) in transwell inserts with or without MFC in the lower well. The presence of MFC stimulated epithelial cell growth, induced alveolar morphogenesis, and enhanced casein accumulation, a marker of the functional differentiation of MEC, but did not induce ductal morphogenesis. Potent mitogenic, morphogenic, and lactogenic effects were observed when the MFC were cultured either on plastic or within a layer of RBM. Although most MFC maintained on plastic died after 1 wk in serum-free medium, fibroblast survival was enhanced significantly when the MFC were cultured within the RBM. Taken together, this in vitro model effectively reconstitutes a physiologically relevant three-dimensional microenvironment for MEC and MFC, and seems ideal for studying the locally derived factors that regulate the developmental fate of the epithelial and fibroblast compartments of the mammary gland.

Selective changes in EGF receptor expression and function during the proliferation, differentiation and apoptosis of mammary epithelial cells.

Epidermal growth factor (EGF) is a multifunctional regulator of mammary epithelial cells (MEC) that transduces its signals through the EGF receptor (EGFR). To clarify the role of the EGFR in the mammary gland, EGFR expression, localization and function were examined during different developmental stages in rats. Immunoblot analysis demonstrated high levels of EGFR during puberty, pregnancy and involution as well as at sexual maturity, and low levels throughout lactation. An immunohistochemical assay was used to show that EGFR was distinctly expressed in a variety of cell types throughout mammary glands from virgin rats and rats during pregnancy and involution, and was down-regulated in all cell types throughout lactation. To examine the relationship between EGFR expression and function, primary MEC were cultured under conditions that induced physiologically relevant growth, morphogenesis and lactogenesis. Cultured MEC expressed an in vivo-like profile of EGFR. EGFR was high in immature MEC, down-regulated in functionally differentiated MEC, and then up-regulated in terminally differentiated and apoptotic MEC. An inhibitor of the tyrosine kinase domain of EGFR was used to demonstrate that EGFR signaling was required for growth and differentiation of immature MEC, and for survival of terminally differentiated MEC, but not for maintaining functional differentiation.

Multiple differentiation pathways of rat mammary stromal cells in vitro: acquisition of a fibroblast, adipocyte or endothelial phenotype is dependent on hormonal and extracellular matrix stimulation.

It has previously been shown that mammary stromal cells possess the ability to maintain a fibroblast-like phenotype or differentiate in vitro into mature adipocytes in a hormone-dependent manner. This paper reports that rat mammary stromal cells can also differentiate into capillary-like structures in vitro when cultured on a reconstituted basement membrane (RBM). The differentiation potential of mammary stromal cells was compared with that of human umbilical vein endothelial cells (HUVEC) and 3T3-L1 preadipocytes. When cultured on plastic, mammary stromal cells, 3T3-L1 and HUVEC maintained a fibroblast-like phenotype. Mammary stromal cells and 3T3-L1, but not HUVEC, differentiated into mature adipocytes when cultured in adipogenic medium. When plated on reconstituted basement membrane, all three cell types began to migrate and organize themselves into an interconnected capillary network. By 18-20 h, mammary stromal cells organized into complex, highly branched capillary-like tubules whereas 3T3-L1 cells and HUVEC formed more simple structures. Cross-sectional analysis demonstrated the presence of an internal lumen. Mammary stromal cells were unique in their ability to progressively develop into a three-dimensional, highly branched network invading the RBM surface. The network formation was enhanced by the presence of vascular endothelial growth factor (VEGF) and was inhibited by the anti-angiogenic drug suramin. Western blotting analysis demonstrated the presence of the endothelial-specific marker flk-1, as well as the presence of the tight-junction-associated protein ZO-1. Mammary stromal cell differentiation into capillary structures was not a terminal state, since these cells were still able to differentiate into adipocytes when exposed to adipogenic medium. These findings suggest that mammary stromal cells differentiate into fibroblasts, adipocytes or vascular structures in a hormone- and substatum-dependent manner, and may explain the dramatic changes in stromal composition during both normal mammary gland development and tumorigenesis.

Adipocyte-epithelial interactions regulate the in vitro development of normal mammary epithelial cells.

Mammary epithelial organoids (MEO), isolated from pubescent rats, were cultured within a reconstituted basement membrane in transwell inserts, in the presence or absence of mature mammary adipocytes in the lower well. This system allowed for free medium exchange between the two compartments, without direct cell-to-cell contact. When cultured in serum-free medium supplemented with insulin, prolactin, hydrocortisone, progesterone, and various epidermal growth factor (EGF) concentrations, mammary adipocytes did not affect epithelial cell growth, but enhanced epithelial differentiation. Casein and lipid accumulations were monitored as indicators of functional differentiation of MEO. Mammary adipocytes significantly enhanced casein and lipid accumulation within the MEO, independently of EGF concentration. Furthermore, adipocytes induced MEO to preferentially undergo alveolar morphogenesis, inhibited squamous outgrowth, and increased lumen size. These findings demonstrate that morphological and functional differentiation of mammary epithelial cells is profoundly enhanced by the adipose stroma and that these effects are mediated by diffusible paracrine factors. This new model can be exploited in future studies to define the mechanisms whereby hormones and growth factors regulate mammary gland development and carcinogenesis. Moreover, it could complement in vivo reconstitution/transplantation studies, which are currently employed to evaluate the role of specific gene deletions in the regulation of mammary development.

The epidermal growth factor receptor is not required for tumor necrosis factor-alpha action in normal mammary epithelial cells.

Our laboratory has shown that tumor necrosis factor-alpha (TNF alpha) can regulate normal mammary epithelial cell (MEC) growth, morphogenesis, and, under certain circumstances, functional differentiation in a manner similar to epidermal growth factor (EGF). As TNF alpha has been shown to up-regulate EGF receptor (EGFR) expression and function in other systems, the present studies were undertaken to determine whether TNF alpha action in MEC was indirect through stimulation of the EGFR. An inhibitor of EGFR tyrosine kinase activity, PD158780, failed to block proliferation induced by 40 ng/ml TNF alpha and only partially inhibited growth in response to 2 ng/ml TNF alpha. PD158780 was also unable to suppress the extensive morphological development induced by either TNF alpha concentration. In contrast, the effects of TNF alpha and PD158780 on functional differentiation (i.e. casein accumulation) were time dependent. When measured on day 7 after 48 h of treatment, casein accumulation was unaffected by either concentration of TNF alpha or by PD158780. When assessed on day 21 after 16 days of treatment, however, casein levels were decreased by 40 ng/ml TNF alpha and increased by PD158780. Significantly, this PD158780-induced increase in casein was not observed in MEC that had been treated with both PD158780 and TNF alpha. These results thus suggest that EGFR tyrosine kinase activity is not necessary for TNF alpha action in normal MEC.

Three-dimensional mammary primary culture model systems.

Model systems have been developed to investigate the complex and coordinated regulation of mammary gland development and transformation. Primary cultures, using newly isolated cells or tissue, are optimal for such studies since, in comparison to immortalized cell lines, the normal signal transduction pathways are presumed to be intact. Three such models are described, including whole organ culture, mammary epithelial cell (MEC) organoids, and MEC-stromal cocultures. Studies using whole-organ culture have the advantage that the normal glandular architecture remains intact, the MEC can undergo lobuloalveolar development and express milk proteins in a hormone dependent manner, and, following hormonal withdrawal, undergo involution. Moreover, transformation of the MEC is readily accomplished. Culture of isolated MEC organoids within an EHS-derived reconstituted basement membrane permits extensive proliferation, branching end bud and alveolar morphogenesis, and accumulation of milk protein and lipid in a physiologically relevant hormone- and growth factor-dependent manner. This model can thus be utilized to investigate the mechanism by which various modulators exert their direct effects on the epithelium. Finally, in view of compelling evidence for stromal-epithelial interactions during normal mammary gland development, and potentially also during the development of malignancy, models in which MEC can be cocultured with enriched populations of stroma offer considerable potential as a tool to understand the nature and mechanisms of the interactions that occur during the various developmental states, and how such interactions may go awry during carcinogenesis.

Prolactin and epidermal growth factor regulation of the proliferation, morphogenesis, and functional differentiation of normal rat mammary epithelial cells in three dimensional primary culture.

The epithelial cell-specific effects of prolactin and epidermal growth factor (EGF) on the development of normal rat mammary epithelial cells (MEC) were evaluated using a three dimensional primary culture model developed in our laboratory. Non-milk-producing MEC were isolated as spherical end bud-like mammary epithelial organoids (MEO) from pubescent virgin female rats. The cultured MEO developed into elaborate multilobular and lobuloductal alveolar organoids composed of cytologically and functionally differentiated MEC. Prolactin (0.01-10 micrograms/ml) and EGF (1-100 ng/ml) were each required for induction of cell growth, extensive alveolar, as well as multilobular branching morphogenesis, and casein accumulation. MEO cultured without prolactin for 14 days remained sensitive to the mitogenic, morphogenic, and lactogenic effects of prolactin upon subsequent exposure. Similarly, cells cultured in the absence of EGF remained sensitive to the mitogenic and lactogenic effects of EGF, but were less responsive to its morphogenic effects when it was added on day 14 of a 21-day culture period. If exposure to prolactin was terminated after the first week, the magnitude of the mitogenic and lactogenic effects, but not the morphogenic response was decreased. Removal of EGF on day 7 also reduced the mitogenic response, but did not have any effect on the magnitude of the lactogenic or morphogenic responses. These studies demonstrate that physiologically relevant development of normal MEC can be induced in culture and that this model system can be used to study the mechanisms by which prolactin and EGF regulate the complex developmental pathways operative in the mammary gland.

Hydrocortisone and progesterone regulation of the proliferation, morphogenesis, and functional differentiation of normal rat mammary epithelial cells in three dimensional primary culture.

The mechanisms of action of, and resistance to, the steroidal regulators of normal mammary epithelial and breast cancer cell development are only partially understood. A major obstacle to research progress has been the difficulty in supporting physiologically relevant development of normal mammary epithelial cells (MEC) under defined serum-free conditions. A primary culture system was developed in our laboratory that permits nonfunctional rat MEC to undergo extensive proliferation, functional differentiation, as well as multilobular and lobuloductal branching alveolar morphogenesis. In the studies reported here, the contributions of hydrocortisone and progesterone during the coordinate induction of cellular proliferation, organoid morphogenesis, and functional capacity were assessed. Hydrocortisone (0.1-10 microgram/ml) induced alveolar and multilobular branching morphogenesis, suppressed lobuloductal branching morphogenesis, and enhanced casein accumulation. Hydrocortisone also played a role in maintaining alveolar as well as multilobular branching morphogenesis and casein levels. Progesterone (0.01-1 microgram/ml) induced cellular proliferation as well as multilobular and lobuloductal branching morphogenesis, and suppressed casein accumulation. At a supraphysiological concentration (10 micrograms/ml), progesterone inhibited cell growth, alveolar branching morphogenesis, and casein accumulation. MEC cultured without progesterone for up to 1 week retained the ability to respond when subsequently exposed to this steroid. Reversibility studies suggested that progesterone was required for the induction, but not the maintenance of the mitogenic, morphogenic, and lactogenic effects. This physiologically relevant primary culture system can be used to study the factors that regulate steroid responsiveness as well as the cross-talk between steroid and growth factor receptor signaling pathways in normal MEC and breast cancer cells.

Modulation of normal mammary epithelial cell proliferation, morphogenesis, and functional differentiation by retinoids: a comparison of the retinobenzoic acid derivative RE80 with retinoic acid.

The ability of retinoids to modulate the proliferation as well as the morphological and functional differentiation of normal mammary epithelial cells isolated from pubescent female virgin rats was evaluated in serum-free primary culture. The retinobenzoic acid derivative RE80, present continuously or for only a limited time in culture, inhibited proliferation with an IC50 of less than 10(-10) M. In contrast, all-trans-retinoic acid (RA) inhibited proliferation with an IC50 of approximately 10(-8) M. In addition to effects on proliferation, RE80 and RA stimulated end bud colonies to differentiate to lobular alveolar colonies, inhibited alveolar budding, and suppressed the outgrowth of squamous colonies. Both retinoids also markedly stimulated functional differentiation, as assessed by accumulation of the major milk protein casein, and stimulated the synthesis of a approximately 73- to 74-kilodalton protein identified as a member of the transferrin family. Moreover, both retinoids stimulated cell death in the differentiated cell population. RE80 was approximately 100-fold more potent than RA for all of these effects. These data suggest that several mechanisms may contribute to the chemopreventive and/or therapeutic efficacy of retinoids in breast cancer, including inhibition of proliferation, stimulation of cell death, and/or induction of differentiation.

Interaction of retinoids with steroid and peptide hormones in modulating morphological and functional differentiation of normal rat mammary epithelial cells.

The interaction of the retinoid RE80 with the lactogenic and mammogenic regulators of mammary gland development was investigated using a mammary epithelial cell (MEC) primary culture model in which cells from young virgin rats were cultured within a reconstituted basement membrane using defined serum-free medium. RE80 (10(-10) M) was able to substitute completely for epidermal growth factor and partially for hydrocortisone in stimulating both morphological and functional (casein accumulation) differentiation of the MEC. In contrast, the requirement of PRL for both differentiation processes was absolute. Furthermore, RE80 was found to abrogate the inhibitory effect of progesterone on casein accumulation and to act as an antiprogestin in terms of morphological effects. Under optimal medium conditions, RE80 also inhibited cell proliferation. This inhibition did not require epidermal growth factor, hydrocortisone, progesterone, or PRL, but, unexpectedly, was enhanced in medium deficient in or lacking hydrocortisone. Additionally, RE80 induced the death of differentiated MEC, an effect that was found to require hydrocortisone. These results suggest that retinoids may modulate transcription of the casein gene family, either directly by activation of the binding of retinoic acid receptors to the casein promoter or indirectly by modulation of the effects of other hormones. The antiproliferative effect of retinoid may also be direct or indirect by virtue of down-regulation of the receptors for one of the mitogenic hormones, possibly progesterone.

Phorbol 12-myristate 13-acetate stimulates proliferation and ductal morphogenesis and inhibits functional differentiation of normal rat mammary epithelial cells in primary culture.

The effect of the tumor promoter phorbol 12-myristate 13-acetate (PMA) on proliferation and differentiation of normal mammary epithelial cells from 50-day-old virgin rats was investigated using a model system that allows for full morphological and functional development of the cells. In this model, mammary epithelial cells are grown within a reconstituted basement membrane in a defined serum-free medium. PMA at a concentration of 10(-6) M effected translocation of protein kinase C from cytosol to membrane. At the same concentration, it stimulated cell proliferation both in the presence and absence of EGF, and this stimulation was observed even when PMA exposure was limited to 15 min at the time of each media change. In contrast to the effect on proliferation, PMA at concentrations of 10(-7) and 10(-6) M inhibited functional differentiation as assessed by casein accumulation. Phorbol 12,13-dibutyrate at 10(-6) M also stimulated proliferation and inhibited casein accumulation and was more effective than PMA in both cases. In contrast, the nonactive tumor promoter 4-alpha PMA had no effect on either proliferation or differentiation. One of the most striking effects of PMA was its ability to stimulate an atypical ductal morphogenesis, as manifested by the formation of intricate web-like colonies, and to inhibit the development of the well-differentiated alveolar-like multilobular colonies. PMA was also shown to completely suppress the growth of the squamous-like colonies that develop when EGF is absent or deficient. These effects of phorbol esters in mammary epithelial cells to stimulate proliferation, inhibit functional differentiation, and stimulate the development of ductal colonies are consistent with the suggestion that the signal transduction pathways evoked by PMA could act to stimulate the growth of initiated cells or render normal cells more sensitive to carcinogen.